DNA replication in eukaryotic cells is tightly controlled so that the genome is replicated once and only once per cell cycle. Such a mechanism is extremely important for faithful transmission of genetic information from one generation to the next. Over-replication of chromosomal DNA will result in genetic instability, which is often associated with human diseases, such as cancer. Our long-term objectives are to understand how DNA rereplication is prevented in mammalian cells and how loss of DNA replication control would lead to genome instability and tumorigenesis. One key to the control of DNA replication initiation is the tightly regulated assembly of pre- replication complexes (pre-RCs) at replication origins by the licensing control mechanism. We showed that when the licensing control is compromised, the ATR-mediated checkpoint is activated and plays a critical role in the suppression of DNA rereplication. Our recent studies further demonstrate that DNA repair machineries are actively involved in removing over-replicated DNA regions and repairing rereplication-associated DNA lesions. We propose that both S-phase checkpoint and DNA repair functions are critical for the maintenance of genome stability when the licensing control is impaired. In this proposal, we will investigate the mechanisms underlying the S-phase checkpoint control and DNA repair activities in the suppression of DNA rereplication and in the repair of rereplication-associated DNA lesions. First, we will study the biological importance of the communication between the S-phase checkpoint and the replication licensing control through a direct interaction of checkpoint proteins with the licensing factor Cdt1. Second, we will investigate the role of DNA repair mechanisms in the removal of rereplicated DNA and in the repair of DNA double-strand breaks (DSBs) that arise during DNA rereplication. We will also define the repair pathways which are used to repair DSBs during DNA rereplication. Third, we will probe the role of the Mre11/Rad50/Nbs1 complex in the suppression of DNA rereplication and investigate the mechanisms underlying this function. DNA rereplication would inevitably lead to genome instability, which is an integral aspect of the malignant phenotype. Recent observations that rereplication or unscheduled DNA replication is induced at the initial stages of tumorigenesis highlight the importance of replication control in the prevention of cancer. Understanding how rereplication is prevented in mammalian cells will shed light on the cellular mechanisms which govern genome stability and improve our understanding of cancer etiology. PUBLIC HEALTH RELEVANCE: DNA rereplication or over-replication of chromosomal DNA often contributes to genome instability, which is highly associated with cancer development. Therefore, understanding how DNA rereplication is suppressed in mammalian cells is of great importance for clarifying the mechanisms underlying the prevention of tumorigenesis. The proposed studies will shed light on cancer etiology and will ultimately help develop new therapeutic interventions for human diseases associated with genome instability and cancer.